Bottlebrush Polyethylene Glycol Nanocarriers Translocate across Human Airway Epithelium via Molecular Architecture-Enhanced Endocytosis

Pulmonary drug delivery is critical for the treatment of respiratory diseases. However, the human airway surface presents multiple barriers to efficient drug delivery. Here, we report a bottlebrush poly(ethylene glycol) (PEG-BB) nanocarrier that can translocate across all barriers within the human airway surface. Guided by a molecular theory, we design a PEG-BB molecule consisting of a linear backbone densely grafted by many (∼1000) low molecular weight (∼1000 g/mol) polyethylene glycol (PEG) chains; this results in a highly anisotropic, wormlike nanocarrier featuring a contour length of ∼250 nm, a cross-section of ∼20 nm, and a hydrodynamic diameter of ∼40 nm. Using the classic air–liquid-interface culture system to recapitulate essential biological features of the human airway surface, we show that PEG-BB rapidly penetrates through endogenous airway mucus and periciliary brush layer (mesh size of 20–40 nm) to be internalized by cells across the whole epithelium. By quantifying the cellular uptake of polymeric carriers of various molecular architectures and manipulating cell proliferation and endocytosis pathways, we show that the translocation of PEG-BB across the epithelium is driven by bottlebrush architecture-enhanced endocytosis. Our results demonstrate that large, wormlike bottlebrush PEG polymers, if properly designed, can be used as a carrier for pulmonary and mucosal drug delivery.


Supporting Text
Molecular structure of a bottlebrush polymer in solution.In a bottlebrush polymer, the side chains highly overlap with each other.This results in steric repulsion that extends the side chains radially away from the bottlebrush backbone, forming a cylindrical shape with cross-section illustrated in Fig. S1A.To calculate the cross-section of the bottlebrush or the size of a side chain,  !" , we consider the volume fraction, (), of the side chains at distance  from the bottlebrush backbone.
Within the cylinder-like bottlebrush polymer, the correlation length is related to the linear grafting density 1/ of side chains 2,3 : which increases with the distance  by a power of 1/2 (Fig. S1B).Note that in our bottlebrush polyethylene glycol (PEG-BB) polymer, the grafting distance is very small with  = 0.254 nm, much smaller than the size  = 0.8 nm of a PEG Kuhn monomer 4,5 .Thus, at the length scale  < , the side chains fill the space to form an exclusion zone for solvents, as predicted by previous theory 6 and confirmed by simulation 7 , as well as illustrated by the shadowed circle in Fig. S1A and the dashed line in Fig. S1B.However, the exclusion zone is very small, on the order of the Kuhn monomer size.Thus, we ignore the effects of exclusion zone on the bottlebrush thickness and focus on the region with  > , where the polymer chains interact with solvent molecules.
Substituting eq.(S3) to eq. (S2), the volume fraction profile can be re-written in terms of the distance  from the bottlebrush backbone: The size of a side chain,  !" , can be determined based on mass conservation: B () where  -,!" is the number of Kuhn monomers per side chain.Solving eq.(S4) one obtains: The bottlebrush polymer is effectively a 'fat' linear polymer with an effective Kuhn length about the cross-section of the bottlebrush,  0 ≈ 2 !" .The radius gyration of this 'fat' linear polymer is proportional to the end-to-end distance: where  = 0.4 for a linear polymer in good solvent 8 and  234 =  !"  is the contour length of the bottlebrush backbone with  !" being the number of side chains per bottlebrush.
Since water is a good solvent for PEG ( = 3/5), the size of the side chain [eq.( S6)] can be rewritten as: -,!" # 5 , for good or athermal solvent (S8) This suggests that the side chain size not only increases with the grafting density 1/ but also scales with the polymer MW by a power of 3/4, higher than 3/5 for an unperturbed linear chain in good solvent.
For PEG in water, the size and mass of a Kuhn monomer are, respectively,  = 0.8 nm and  6 = 44 g/mol 4,9 .In our densely grafted PEG-BB, the number of side chains is  !" = 990, the grafting distance is  = 0.254 nm, and the number of Kuhn monomers per side chain is  -,!" =  !" / 6 ≈ 22, in which  !" = 950 g/mol is the molecular weight of a PEG side chain.Substituting these numbers into eqs.(S8) and (S7), one obtains the cross-section of the bottlebrush,  0 ≈ 2 !" ≈ 20 nm, and the size of the bottlebrush,  1 ≈ 36 .The hydrodynamic diameter  7 of a 'fat' linear polymer is related to its radius of gyration  1 by  7 = 1.25 1 ≈ 45 nm (Table 8.4 in ref. 1 ).
Considering that scaling theory ignores prefactors on the order of unity, the predicted value agrees reasonably well with the measured hydrodynamic diameter 37 ± 0.4 nm (Fig. 1F).
For the loosely grafted PEG (PEG-LG), the number of side chains is  !" = 750 and the grafting distance  = 1.524 nm is much larger than the size of a PEG Kuhn monomer.As a result, the side chains are not much overlapped and adopt a nearly unperturbed conformation.The backbone of the bottlebrush polymer is not strained and adopts a self-avoiding random walk with  1 ≈ ( 234 /) #/8 ≈ 33 nm (where  ≈ 1.7 nm for a methyl methacrylate-based polymer 10 and  234 ≈ 1100 nm is the backbone contour length).

Figure S1 .
Figure S1.Cross-section of a bottlebrush polymer in solution.(A) An unperturbed bottlebrush polymer with the thickness  is expected to be larger than the size of the free side chain.The correlation length () increases with the distance  from the bottlebrush backbone.(B) Mesh size profile for an unperturbed bottlebrush polymer.Yet, at a very high grafting density with the distance  between two neighboring grafting sites much smaller than the Kuhn monomer size , the side chains form an exclusion zone (dashed line), as illustrated by the shadowed light blue circle in (A).Logarithmic scales.

Figure S6 .
Figure S6.PEG-BB homogenizes with increased incubation time.(A) Representative fluorescence images of PEG-BB at the apical, middle, and basal focal planes after incubation for 1 hour, 6 hours, and overnight.(B) Fluorescence fraction of PEG-BB at different focal planes after incubation for 1 hour, 6 hours, and overnight.Statistical analysis is performed using one-way ANOVA.n.s., not significant; *, p<0.05.n=5 donors.

Figure S8 .
Figure S8.Uptake of polymers with different molecular architectures by NIH-3T3 fibroblasts.Representative pre-and post-wash images of NIH-3T3 fibroblasts after overnight incubation with (A) 2 MDa dextran, a randomly branched inert molecule, (B) 70 kDa dextran, and (C) 1 MDa PEG-LG.All polymers are added to the medium at the same concentration of 100 μg/ml.Pre-wash: cells are imaged right after incubation without replacing the cell culture medium.Post-wash: cells are imaged after being washed with the fresh culture medium to remove free extracellular molecules.After overnight incubation, FITC-labeled 2 MDa dextran is not uptaken by NIH-3T3 cells, rendering the cell contour circumscribed.70 kDa dextran is negligibly uptaken into NIH-3T3 cells as minimal perinuclear green dots.PEG-LG, which has a comparable molecular weight and hydrodynamic size to PEG-BB, is uptaken into NIH-3T3 cells slightly higher than 70 kDa dextran, showing as perinuclear green fluorescence dots.However, the uptake of PEG-LG is significantly less than PEG-BB.

Figure S9 .
Figure S9.Cell uptake of PEG-BB is inhibited by endocytosis inhibitors.Representative images of NIH-3T3 cells treated with (A) 0.1 μg/ml wortmannin in the medium, (B) 5 μg/ml chlorpromazine in the medium, and (c) 5 μg/ml nystatin in the medium for 4 hours.Then, cells are washed with the prewarmed culture medium and incubated overnight with 100 μg/ml PEG-BB in the culture medium.Pre-wash: cells are imaged right after incubation without replacing the culture medium.Post-wash: cells are imaged after being washed with fresh culture medium to remove free extracellular PEG-BB.After being treated with wortmannin, chlorpromazine, and nystatin to inhibit endocytosis, almost all PEG-BB is unabsorbed and remains extracellular, outlining cell contour in the pre-wash images.After washing, minimal PEG-BB remains intracellular and presents as perinuclear green fluorescence dots in the post-wash images.